ChIP-seq analysis of genomic binding regions of five major transcription factors highlights a central role for ZIC2 in the mouse epiblast stem cell gene regulatory network.

Kazunari Matsuda,Munazah Andrabi,Jeremy M Boss,Tomoyuki Mikami,Shuji Shigenobu,Hideaki Iida,Hisato Kondoh,Shinya Oki,Katsushi Yamaguchi

doi:10.1242/dev.143479

Abstract

To obtain insight into the transcription factor (TF)-dependent regulation of epiblast stem cells (EpiSCs), we performed ChIP-seq analysis of the genomic binding regions of five major TFs. Analysis of in vivo biotinylated ZIC2, OTX2, SOX2, POU5F1 and POU3F1 binding in EpiSCs identified several new features. (1) Megabase-scale genomic domains rich in ZIC2 peaks and genes alternate with those rich in POU3F1 but sparse in genes, reflecting the clustering of regulatory regions that act at short and long-range, which involve binding of ZIC2 and POU3F1, respectively. (2) The enhancers bound by ZIC2 and OTX2 prominently regulate TF genes in EpiSCs. (3) The binding sites for SOX2 and POU5F1 in mouse embryonic stem cells (ESCs) and EpiSCs are divergent, reflecting the shift in the major acting TFs from SOX2/POU5F1 in ESCs to OTX2/ZIC2 in EpiSCs. (4) This shift in the major acting TFs appears to be primed by binding of ZIC2 in ESCs at relevant genomic positions that later function as enhancers following the disengagement of SOX2/POU5F1 from major regulatory functions and subsequent binding by OTX2. These new insights into EpiSC gene regulatory networks gained from this study are highly relevant to early stage embryogenesis.

Highlights

Mammalian somatic cells originate from the epiblast in postimplantation embryos
(2) The enhancers bound by ZIC2 and OTX2 prominently regulate transcription factor (TF) genes in epiblast stem cells (EpiSCs). (3) The binding sites for SOX2 and POU5F1 in mouse embryonic stem cells (ESCs) and EpiSCs are divergent, reflecting the shift in the major acting TFs from SOX2/POU5F1 in ESCs to OTX2/ZIC2 in EpiSCs. (4) This shift in the major acting TFs appears to be primed by binding of ZIC2 in ESCs at relevant genomic positions that later function as enhancers following the disengagement of SOX2/POU5F1 from major regulatory functions and subsequent binding by OTX2
Fundamentals of the ChIP-seq procedure using in vivo biotinylated TFs Expression of in vivo biotinylated TFs ChIP-seq using a biotinylated TF requires the expression of an exogenous TF fused to a biotin ligase recognition peptide (BLRP) that is biotinylated in vivo by co-expressed E. coli-derived biotin ligase (BirA)

Summary

Introduction

EpiSCs can be maintained in feeder-free culture conditions supplemented with activin, which is a Nodal replacement, and bFGF (FGF2) (Brons et al, 2007; Iwafuchi-Doi et al, 2012) They can be directed towards development into a variety of specific somatic lineages by removing activin and/or manipulating other cell signaling systems (Brons et al, 2007; Tesar et al, 2007; Iwafuchi-Doi et al, 2012; Kojima et al, 2014; Matsuda and Kondoh, 2014; Tsakiridis et al, 2014; Li et al, 2015). Simple removal of activin/Nodal signaling directs EpiSCs to develop into anterior neural plate (ANP) cells (Iwafuchi-Doi et al, 2012)

Methods

Results

Conclusion